Impact of dietary administration of Arthrospira platensis free-lipid biomass on growth performance, body composition, redox status, immune responses, and some related genes of pacific whiteleg shrimp, Litopenaeus vannamei

The current study aimed to assess the influence of dietary inclusion of cyanobacterium Arthrospira platensis NIOF17/003 as a dry material and as a free-lipid biomass (FL) on the growth performance, body composition, redox status, immune responses, and gene expression of whiteleg shrimp, Litopenaeus vannamei postlarvae. L. vannamei were fed five different supplemented diets; the first group was fed on an un-supplemented diet as a negative control group (C-N), the second group was fed on a commercial diet supplemented with 2% of A. platensis complete biomass as a positive control group (C-P20), whereas, the three remaining groups were fed on a commercial diet supplemented with graded amounts of FL at 1%, 2%, and 3% (FL10, FL20, and FL30, respectively). The obtained results indicated that the diet containing 1% FL significantly increased the growth performance, efficiency of consumed feed, and survival percentage of L. vannamei compared to both C-N and C-P20 groups. As for the carcass analysis, diets containing A. platensis or its FL at higher levels significantly increased the protein, lipid, and ash content compared to the C-N group. Moreover, the shrimp group fed on C-P20 and FL10 gave significantly stimulated higher digestive enzyme activities compared with C-N. The shrimp fed C-P20 or FL exhibited higher innate immune responses and promoted their redox status profile. Also, the shrimp fed a low FL levels significantly upregulated the expression of both the peroxiredoxin (Prx) and prophenoloxidase (PPO1) genes than those receiving C-N. The current results recommended that dietary supplementation with 1% FL is the most effective treatment in promoting the performance and immunity of whiteleg shrimp.

recommended that dietary supplementation with 1% FL is the most effective treatment in promoting the performance and immunity of whiteleg shrimp.

Introduction
The shrimp farming industry has expanded intensively and has become one of the most important leading global aquaculture sectors [1][2][3].The Pacific whiteleg shrimp, Litopenaeus vannamei, has been the most widely cultivated species of all penaeid shrimp species and contributes to more than 70% of the world's shrimp farming [4,5].To sustain the aquaculture industry worldwide, there are several problems to be resolved, including issues in the aquafeed industry, disease, low survivability, and poor water quality [6][7][8][9].Moreover, the harmful effects of environmental pollution and climate change are key factors limiting the sustainability of aquaculture, fisheries, aquatic habitats, and aquatic organisms [10][11][12].
The shrimp feed industry has expanded by implementing a variety of strategies to deal with the global expansion in shrimp farming [7].One of the most significant areas among these strategies is feed additive supplementation, which has become extremely important for numerous shrimp species as growth enhancers, immune stimulants, and a substitute approach for combating disease resistance [13].
The basic factors evaluating the quality of shrimp feed additives are growth performances, feed utilization indicators, biochemical composition, immune-related gene expressions, and immunological indices [14][15][16].The immune system of shrimp is based primarily on innate immunity and includes cellular and humoral, enzymatic and non-enzymatic, and antioxidant effectors.Those are implemented by cellular antioxidant agents that identify invasive pathogens and activate different defense mechanisms to eliminate infections [16].
There are several forms of A. platensis supplementation in the diets of shrimp L. vannamei, such as dry powder form [33], whole-liquid extract form [34], nanoparticle form [13], derivative extract form [35] and lipid-free biomass.Each form has advantages and disadvantages.On the other hand, the procedure of addition to the diet is a key element to the success of the inclusion process.A. platensis contains up to 15.4% lipids [36].Thus, high levels of A. platensis in the shrimp diet may result in increased excessive lipid accumulation and oxidative stress [37].Therefore, it is essential to apply advanced procedures to eliminate the A. platensis lipid content to realize the advantages of adding A. platensis to shrimp feed on growth and health.
In our previous works, the A. platensis lipid-free biomass (FL) was successively evaluated as feed for marine rotifer, Brachionus plicatilis, production, and removing ammonia (phytoremediation) from aquaculture effluents [38].The current study aims to evaluate the effect of A. platensis free-lipid biomass, FL, the biodiesel byproduct) as a dietary supplement on growth performance, feed utilization, and biochemical composition of postlarvae of whiteleg shrimp, were frequently cleaned during the experimental period, and the water turnover rate for each pond was around 10% for each pond per day by intake and output flow rates via the pond system.

Growth performance and feed utilization indices
The L. vannamei weights (g) were recorded at the beginning of the feeding trial (0.05 ± 0.02 g) and every 15 days afterward.At the end of the feeding trial, following a period of starvation, shrimp were counted and individually sampled for length and weight.Obtained data were used to calculate the survival rate, weight gain (WG,g), survival rate (SR,%), specific growth rate (SGR%/day), feed conversion ratio (FCR), feed efficiency ratio (FER), protein intake (PI, g), and protein efficiency ratio (PER) parameters according to the following Eqs (1-7): Weight Gain ðWG; gÞ ¼ Final body weight ðgÞ À Initial body weight ðgÞ ð1Þ

Body chemical analysis
At the end of the experiment, five shrimp from each replicate were collected to estimate the shrimp's whole-body proximate composition.Shrimp were randomly chosen, euthanized, homogenized in a blender, oven-dried, powdered, and preserved at -20˚C for further investigations.The biochemical composition percentages (crude protein, crude lipid, ash, and dry matter) were determined as previously described [41].

Immunological indices
From each replicate, five shrimp, following 24 hours of starvation, were randomly selected and rinsed with sterile seawater for a few seconds.Shrimp tissue samples were dissected, weighed, frozen in liquid nitrogen, and stored at-80˚C until use.For lysozyme, antioxidants, and digestive enzyme assays, the shrimp tissue samples were homogenized, after adding PBS (pH 7.4), centrifuge (20 min, 2,000-3,000 rpm), and the supernatant was carefully collected.2.6.1.Lysozyme activity assay.Serum lysozyme activity was assayed by Lysozyme (LZM) ELISA Kit (Cat NO.:SL0050FI, SunLong Biotech Co., LTD, China).During incubation of the lysozyme sample and Micrococcus lysodeikticus cells as the substrate, the reaction was followed by monitoring the reduction in absorbance reading at 450 nm wavelength following the manufacturer guidelines.

Immune-related gene expressions
At the end of the feeding trial, three equal pools of independent samples of each five L. vannamei shrimp (whole animals) were collected, washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , 1.46 mM KH 2 PO 4 , and pH 7.4), and stored in RNA later 1 reagent (Sigma-Aldrich 1 ; 1w:5v) at-20˚C as ascribed by the procedure of Goncalves et al. [50].The total RNA extraction and quantitative real-time PCR followed the method of Aguilera-Rivera et al. [51].Briefly, the TRIzol reagents protocol (TRIzol © ; Life Technologies) was applied to extract the total amount of RNA then the obtained extraction was quantified at 260 and 280 nm using a NanoDrop spectrophotometer (Thermo Scientific).The RT2 First Strand Kit, which includes a highly successful genomic DNA removal step before reverse transcription, was used during the RNA extraction process to prevent DNA contamination.cDNA was produced in a 10-μL estimated volume including 4 μg of the total extracted RNA, 10 × RT buffer, 10 mM dNTP, 10× random RT primers and U reverse transcriptase (Enhanced Avian RT First Strand Synthesis; Sigma-Aldrich © ).The first strand cDNA was generated at 59˚C for 50 min.Then, the designed primers designated in this experiment, including the Peroxiredoxin (Prx), Prophenoloxidase (PPO1), p53-like protein isoform delta (p53), and hemocyanin subunit L5 (L5H) genes, were presented in Table 2 and prepared for q-RT-PCR estimation, which was conveyed into a fluorometric iQ5 thermocycler (Bio-Rad 1 ) following the Aguilera-Rivera et al. [51] guidelines and applying the gene β-actin as the housekeeping gene [52].
The applied primers of the housekeeping and target genes were designed from the conserved sequences of each gene in Genbank with Primer 5.0 software.The expression level of each gene was estimated and calculated using the 2-ΔΔ C t as ascribed by Livak and Schmittgen [53], where C t is the value corresponding to the number of cycles in which the fluorescence was created.Each real-time PCR reaction (including cDNA synthesis) was repeated triplicate times to ensure the accuracy of the obtained results.Moreover, the qPCR values were log 2 transformed to achieve normality and diminish data variability.Besides, the PCR efficiency for each sample was derived from the slope of the regression line fitted to a subset of baseline-corrected data points in the log-linear phase using LinRegPCR following Ramakers et al., procedure [54].

Statistical analysis
The current feeding trial results were presented (n = 5) as the means ± standard deviation (SD).Before the data were analyzed, the normality and homogeneity assumptions were conducted and the results (%) were arc-sin transformed [55].The statistical procedure was performed using the IBM SPSS (IBM, v.

Biochemical composition of A. platensis free-lipid biomass (FL)
The chemical analyses of the free-lipid biomass of A. platensis are shown in Fig 1 .Biochemical composition (% of DW) of protein, lipid, carbohydrates, and ash of A. platensis free-lipid biomass applied in the current experiment as FL (FL 10 , FL 20 , and FL 30 ) was 66.7%, 0.0%, 16.29%, and 7.93%, respectively.highest shrimp lysozyme activity (3.65 μg mL −1 ) was recorded in the shrimp group treated with a low level of FL (FL 10 ) (Fig 5).Shrimp provided with a complete or free lipid of A. platensis biomass-supplemented diet had significantly higher superoxide-dismutase (SOD) levels than shrimp fed a free basal diet (Fig 5).The highest levels of SOD were found in the C-P 20 and FL 30 groups, followed by the FL 10 and FL 20 groups, respectively.The effects of a supplemented shrimp diet with the complete or free lipid of A. platensis biomass on malonaldehyde (MDA) were significantly lower when compared to the C-N group (Fig 5).Specifically, the shrimp group fed complete A. platensis biomass exhibited the lowest MDA level.In contrast, there were no significant differences in catalase activity between all shrimp experimental groups.At the end of the experiment, all experimental treatments substantially altered digestive enzyme activity (lipase and amylase) (Fig 5).Shrimps fed diets containing complete or free lipid A. platensis had higher levels of amylase and lipase than shrimps fed diets without A. platensis supplementation.Specifically, shrimps fed the C-P 20 diet had the highest levels of digestive enzymes, lipase, and amylase levels gradually increased with increasing FL content in the diets but remained lower than in the shrimp group fed the C-P 20 diet (Fig 5).https://doi.org/10.1371/journal.pone.0300748.g003

Discussion
Over the past decade, numerous researchers have investigated the benefits of using microalgae in whiteleg shrimp cultivation [37,57,58].Although replacing fishmeal with alternative lowercost protein sources has been examined [59,60], using biofuel production by-products as a protein and carbohydrate source or even as feed additives for L. vannamei, has not been extensively investigated.A previous report on microalgae administration found that A. platensis blended with probiotics may promote the growth biometric indices and health status of whiteleg shrimp [61].This current investigation was in line with our results that showed supplemented shrimp diets with varying levels of FL significantly promoted growth performance A similar trend was observed in feed efficiency measurements, where the FL 10 group significantly enhanced feed efficiency when compared to other treated or untreated groups.These findings imply that A. platensis or its by-product-supplied meal has high quantities of protein, which may have a favorable influence on whiteleg shrimp growth and feed efficiency indices when compared to the group fed an algal-free diet.Furthermore, our findings using A. platensis by-product are consistent with the findings of Cuzon et al. [62], who included an 8% lipidfree fraction of A. platensis meal and demonstrated higher growth and survival in Penaeus japonicus.In another report, Nakagawa and Gomez-DI ´az [63] reported a marked enhancement in the performance, survival percentage, pigmentation level, and protein utilization of giant freshwater shrimp (Macrobrachium rosenbergii) fed diets supplementing with 5-10% whole A. platensis meal and attributed the improvements to protein assimilation promotion.In addition, the inclusion of 9% defatted microalgae Nannochloropsis or Thalassiosira weissflogii meals resulted in higher growth performance of L. vannamei [64].
Lipid free algae would have a higher concentration of protein leading to less interference in terms of digestion and assimilation of released amino acids.This is a likley explanation for the superior performance of the FL fed groups over the other treatments evaluated.It is known that shrimp L. vannamei have an inferior ability to procees fats and oils in the diet due to a reduced emusfication capacity of the hepatopancreas compared to fish [65].Most aquatic species have an advanced biliary circulation and entero-hepatic system with the release of bile salts, unlike shrimp.Lipid digestion in shrimp is mainly an intracellular activity in the hepatopancreas epithelium, from which lipids are conveyed to the target tissues and organs by the haemolymph as carrier lipoproteins.The formation and absorption of lipid micelles from the lumen of the hepatopancreas tubuli is therefore a constraint in the lipid digestion and assimilation process [66].Recently, Namaei Kohal et al. [67] reported that most growth indicators, including final weight, specific growth rate, and average daily growth rate, were considerably higher in red cherry shrimp (Neocaridina davidi) fed diets supplemented with 10% Arthrospira platensis.The improvements in growth and efficiency of consumed diet in shrimp fed 1% FL were shown to be connected to that of a meal supplemented with microalgae by-product proven to be a rich source of carotenoids [68] and was regarded as an appropriate and safe feed additive for L. vannamei.Besides, previous reports indicated that bioactive compounds (such as growth hormones, nucleotides, vitamins, and minerals, free amino acids and fatty acids, pigments, and molecules up-regulating gene expression) in diets containing A. platensis might constitute effective agents to promote the functionality of the product and also improve feed consumption by shrimp due to gustatory and olfactory properties [69,70].
The current study reported that feeding A. platensis or higher levels of its byproducts to whiteleg shrimp stimulated the activity of digestive enzymes, including α-amylase (carbohydrate digestion).However, while lipase level was significantly higher in all dietary treatments that included FL compared to the C-N group, we discovered that enzyme activity peaked at the medium level of FL (2%), and then declined with increasing levels of FL (up to 3%) in the diet unexpectedly and inexplicably.This finding is inconsistent with reports by Namaei Kohal et al. [67].It might be attributed to the fact that shrimp fed complete A. platensis or higher doses of FL have the potential to promote the recycling process, which is thought to be a result of both compartmentalizations produced by the presence of the peritrophic membrane and fluid movement in the midgut lumen [71].Also, this theory predicts that an increase in protein or starch in the diet generated by A. platensis supplementation would result in the displacement of the corresponding digestive enzymes, leading to a larger recovery of these enzymes in the feces [72].
The shrimp body content in protein and lipids fed complete or by-product biomass of A. platensis increased in parallel with increasing dietary inclusion levels.The A. platensis based diet's palatability might enhance feed intake, which subsequently increases body carcass composition [32,73].The observed findings were found to be consistent with Radhakrishnan et al. [73] in M. rosenbergii fed diets containing higher levels of A. platensis.Conversely, Namaei Kohal et al. [67] demonstrated that the protein content increased with dietary A. platensis levels up to 10%, but fat content was reduced with rising Spirulina levels in the caridean red cherry shrimp (Neocaridina davidi).Whereas, Namaei Kohal et al. [67] demonstrated that protein content increased with dietary A. platensis levels up to 10%, but fat content reduced with rising A. platensis levels in the caridean red cherry shrimp N. davidi.The difference in our study results and other research findings may be attributed to the applied microalgal species and their protein and fat content, the shrimp species, the application technique, and diet palatability.
As a crustacean, shrimp lack adaptive immunity, hence their health is mostly dependent on non-specific immune functions [74].Superoxidase dismutase (SOD) and lysozyme are enzymes that neutralized cellular free radicals and collapse pathogenic bacterial cell walls, respectively [75].Furthermore, an increase in MDA levels indicates an increase in free radical production, hence it is widely applied as a biomarker of oxidative stress [76].When compared to the C-N group, shrimp fed the complete or by-product of A. platensis had higher SOD activity.These findings correlated with serum lysozyme activity, where shrimp fed low levels of A. platensis by-products (FL 10 ) had much higher levels than the other enriched treatments and the C-N group.Conversely, MDA levels were significantly lower in all shrimp groups fed A. platensis complete biomass or by-products as compared to the C-N group.These findings are consistent with prior research that indicated shrimp fed A. platensis supplemented diets had improved non-specific immune responses, as well as enhanced redox status [70,77,78].
It is commonly known that in addition to phycocyanin, A. platensis or its by-products includes several bioactive molecules specifically, carotenoids and xanthophyll molecules, which have multiple double bonds that bind with free radicals and regulate inflammatory pathways [79].Furthermore, Khan et al. [80] demonstrated that different Spirulina preparations alter the immune system through increasing macrophage phagocytic activity, promoting antibody and cytokine production, increasing NK cell accumulation in tissue, and activation and migration of T and B cells.
The peroxiredoxin (PRX) class of proteins are thiol-specific antioxidants found in all eukaryotes and prokaryotes [81].These proteins serve a critical role in protecting shrimp against oxidative stressors when exposed to physical, chemical, or biological stress, which causes acute oxygen deprivation and irregular metabolic pathways, leading to the production of excessive quantities of free radicals [82].Moreover, the transcription of the PPO1 gene in L. vannamei was discovered to be associated with the maturation of crystal cells, which contain the enzymes required for humoral melanization, which would be linked with a variety of immunological responses [83].In terms of the underlying mechanisms by which A. platensis has a beneficial action on shrimp, it was observed that shrimp hemocytes incubated in Spirulina dried powder (1 mg per mL) activated innate immunity, as evidenced by the recognition and binding of a recombinant protein of lipopolysaccharide and 1,3-β-glucan binding protein (LGBP), degranulation of haemocytes, a reduction in the percentage of large cells, increases in phenoloxidase (PO) and serine proteinase activities, activated superoxide anion levels, and upregulated LGBP gene transcript [77].In this experiment, shrimp-fed diets containing a low level of LF (10 g kg -1 diet) exhibited significant upregulation of both the Prx and PPO1 gene transcripts when compared to other groups.Thus, more research is required to determine if dietary FL may substantially stimulate immunological or antioxidant-related metabolites in L. vannamei.

Conclusion
Several forms of microalga A. platensis supplementation, and/or their extracts, have been applied in the diets of shrimp L. vanname.However, this study revealed that free lipid biomass from A. platensis, compared to the whole-dry weight form, might improve whiteleg shrimp performance, chemical body composition, antioxidant activity, and immunological responses.Diets supplemented with a 10 g/kg diet of A. platensis free-lipid-biomass had the higher shrimp growth rate and superior feed efficiency, moreover, they also achieved the largest improvement on the innate immune response.
23) Statistics Software, by the one-way ANOVA followed by the Tukey's range test, at a significant level of P � 0.05.Finally, Figures were prepared by Graph Pad (Prism 8) Statistics Software [56].

Figs 2 and 3
Figs 2 and 3 shows the influences of dietary supplementations of A. platensis (complete dry weight or FL) on the survival, performance, and feed utilization of the juveniles of L. vannamei, respectively.No statistically significant differences (p � 0.05) in survival rates between all treated and non-treated groups were noted (Fig2).Overall, the 1% FL dietary supplementation significantly improved FBW, WG, SGR, FER, PER, PI, and significantly reduced FCR, compared to the shrimp group fed an un-supplemented diet and all other experimented diets (Figs2 and 3).

Fig 4 Fig 1 .
Fig 4 shows the whole-body biochemical composition of the shrimp fed the different dietary treatments.All shrimp treated with C-P 20 , FL 20 , and FL 30 showed significantly (p � 0.05)

Fig 5
Fig 5  illustrates the influences of experimental diets supplemented with graded amounts of complete or free lipids of A. platensis biomass on immunological responses, redox status, and digestive enzyme secretions of L. vannamei.The innate immune response results showed that there were significant differences (p < 0.05) in lysozyme activities of shrimp groups fed diets supplemented with A. platensis (C-P 20, FL 10 , FL 20 , and FL 30 ) compared to the C-N group.The

Fig 2 .
Fig 2. Influence of experimental diets on growth performance indices of shrimp L. vannamei.C-N: control diet (negative control), C-P 20 : control diet supplemented with 20 g kg −1 of A. platensis complete biomass (positive control), FL 10 , FL 20 , and FL 30 : diets supplemented with 10, 20, and 30 g kg −1 of A. platensis free-lipid biomass.Data were represented as means ± SD.Different letters in each column indicate significant differences (p < 0.05).The absence of letters in each column means that there are no significant differences.https://doi.org/10.1371/journal.pone.0300748.g002

Fig 3 .
Fig 3. Influence of experimental diets on feed utilization indices of shrimp L. vannamei.C-N: control diet (negative control); C-P 20 : control diet supplemented with 20 g kg −1 of A. platensis complete biomass (positive control); FL 10 , FL 20 , and FL 30 : diets supplemented with 10, 20, and 30 g kg −1 of A. platensis free-lipid biomass.Data were represented as means ± SD.Different letters in each column indicate significant differences (p < 0.05).

Fig 6
Fig 6  shows that the mRNA expression of Prx and PPO1 genes was significantly (p < 0.05) influenced by dietary treatments, except for p53 and L5H genes, which were not significantly altered.The expression of the Prx gene was highly upregulated in the treated group given an FL 10-based diet, while FL 20 , FL 30, and C-P 20 insignificantly affected the study gene when compared to the shrimp group fed diets without A. platensis addition (C-N).In contrast, feeding shrimp with two forms of A. platensis based diets significantly increased the expression level of the PPO1 when compared to shrimp fed just a free SP diet.The shrimp fed FL 10 (a low amount of A. platensis free lipid biomass) exhibited the highest expression of the PPO1 gene, followed by the C-P 20 , FL 20 , and FL 30 groups.

Fig 5 .
Fig 5. Influences of experimental diets on immunological indices of shrimp L. vannamei.C-N: control diet (negative control); C-P 20 : control diet supplemented with 20 g kg −1 of A. platensis complete biomass (positive control); FL 10 , FL 20 , and FL 30 : diets supplemented with 10, 20, and 30 g kg −1 of A. platensis free-lipid biomass.Data were represented as means ± SD.Different letters in each column indicate significant differences (p < 0.05).The absence of letters in each column means that there are no significant differences.https://doi.org/10.1371/journal.pone.0300748.g005

Fig 6 .
Fig 6.Influence of experimental diets on mRNA expression of shrimp L. vannamei.C-N: Control diet (negative control); C-P 20 : control diet supplemented with 20 g kg −1 of A. platensis complete biomass (positive control); FL 10 , FL 20 , and FL 30 : diets supplemented with 10, 20, and 30 g kg −1 of A. platensis free-lipid biomass.Data were represented as means ± SD.Different letters in each column indicate significant differences (p < 0.05).The absence of letters in each column means that there are no significant differences.https://doi.org/10.1371/journal.pone.0300748.g006

Table 1 . The formulation and chemical composition of the basal diet (dry matter basis).
Binder was providing (Guar gum) was imported from Pakistan and supplied by Hoa chat Can Tho comp, Vietnam.
c VMC Group Vietnam.dMaharashtra Solvent extraction LTD India.e An Giang Agriculture and foods import-export joint stock company.f